RR4

Question 1

How does liquid chromatography present an advantage when isolating transcription factors?

Answer 1

You can mix the factors back together after invitro inspection

Question 2

Explain how we can label an RNA during its’ synthesis

Answer 2

1) When you cut DNA, introduce protein
2) Protein will start transcription, synthesizing an RNA porduct
3) Label RNA transcript synthesized in reaction using rNTPs
4) OR use unlabelled RNA, instead use a labelled probe to see how much RNA was synthesized

Question 3

How does liquid chromatography for the isolation of TFs work?

Answer 3

Nuclear extract is separated into columns - combinations of specific fractions allowed transcription in RNA pol II
Can see the columns that allow the transcription would have the important transcription factors

Question 4

What is the TBP?

Answer 4

The TATA box binding protein - makes up TF2D
- binds to DNA minor groove, kinks DNA and distorts it
- Critical in TATA box binding and in vitro (no TATA)
- Involved in class I and class III base transcription

Question 5

What are 5 major transcription factors?

Answer 5

• TF2A
• TF2B
• TF2H
• TF2D
• TF2E

Question 6

What is a TAF?

Answer 6

“TBP associated factors”
make up all subunits of TF2D
Some TAFs interact with elements around start site even when there is no TATA

Question 7

What are the steps of pre initiation?

Answer 7

1) TF2D binds to promoter, interacts tighlty with DNA, summons TF2A and TF2B
2) TF2A, TF2B, and TF2D stabilize the complex - makes ‘upstream promoter complex’
3) RNA pol II can react with complex - it is bound to TF2F, it binds to the UPC
4) This creates a “core pre-initiation complex” - gene promoter can now activate
5) TF2E and TF2H come into complex, completely stabilizing it
6) With ATP and dATP, DNA will open, RNA pol II can now interact with the strand, and elongation begins (with rNTPs)

Question 8

What does TF2H do?

Answer 8

• Contain ATP dependent activity that will melt DNA, opening the transcription bubble
• Contains 9 polypeptides involved in repair activities
• Contains helicases involved in xeroderma pigmentosum

Question 9

What is TF2H coupled with?

Answer 9

Transcription coupled repair

Question 10

Explain Xeroderma pigmentosum

Answer 10

• Faulty NER
  Cells can’t repair after sun damage due to TF2H issues

Question 11

What matters in speed of repair of genome lesions? What are they linked with?

Answer 11

• TF2H is higher in some regions - coincide with the regions of transcriptional start sites- as well as regions of quick repair

Question 12

What TFs make up the UPC?

Answer 12

TF2A, TF2D, and TF2B
Stabilizes complex